There is a need for sensitive apparatus and methods for the detection of individual molecules or particles. It is particularly important in medical and biological research to be able to measure the concentration, number or position of individual particles such as bacteria, viruses, and DNA fragments which are intrinsically fluorescent or can be labeled with fluorescent markers or probes.
In the quest for enhanced sensitivity, Hirschfeld used evanescent-wave excitation to detect an antibody molecule labeled with 80 fluoresceins adsorbed on a glass slide..sup.1 Using a flowing sample, Dovichi et al..sup.2 achieved a detection limit of 22,000 rhodamine 6G molecules in a 1 s integration time, and Nguyen et al..sup.3 extended this limit to 800 molecules with hydrodynamically-focused flows. Mathies and Stryer.sup.4 pointed out the limits imposed by photodestruction and detected three molecules of B-phycoerythrin (PE) in a probe volume of 10 pL. Recently, Nguyen et al. observed bursts of fluorescence when a 10.sup.-12 M solution of PE was flowed through a focused laser beam, and they interpreted these bursts as being due to the passage of individual molecules.sup.5. To detect single molecule fluorescence bursts, one must ensure that the probability of observing emission from two molecules simultaneously in the beam is negligible. In the distribution function, the probability of detecting zero counts during a given time interval from the fluorescent sample should differ from that of the solvent by less than 10%. A convenient test is that the mean count rate in the sample should increase by less than 10% compared to the blank. In the experiments of Nguyen et al., the most probable count rate with PE is double that in the blank and their probability for single occupancy (0.34) gives a double occupancy probability of 0.11. This indicates that Nguyen et al. were observing bursts of fluorescence due to the simultaneous presence of two or more molecules in the imaged volume rather than the presence of single molecules. FNT .sup.1 Hirschfeld, T. (1976) Appl. Optics 15, 2965-2966. FNT .sup.2 Dovichi, N. J., Martin, J. C., Jett, J. H., Trkula, M. & Keller, R. A. (1984) Anal. Chem 56, 348-354. FNT .sup.3 Nguyen, D., Keller, R. & Trkula, M. (1987) J. Opt. Soc. Am. 4, 138-143. FNT .sup.4 Mathies, R. A. & Stryer, L. (1986) in Fluorescence in the Biological Sciences, eds. Taylor, D. L., Waggoner, A. S., Lanni, F., Murphy, R. F. & Birge, R. (Alan R. Liss, Inc., New York), 129-140. FNT .sup.5 Nguyen, D. C., Keller, R. A., Jett, J. H. & Martin, J. C. (1987) Anal. Chem 59, 2158-61.
The prior art does not provide a method and apparatus which can provide rapid ultrasensitive detection and counting of fluorescent particles down to the single molecule limit, nor does the prior art provide a method for determining the optimal conditions to obtain this high detection sensitivity.